Cellular microvesicles (MVs) are a category of biological vesicles with a lipid bi-layer membrane, ranging between 10-500 nm in size. They were first reported as early as in year 1967 and named “platelet dust” since they were derived from platelets, contain vesicles and have a role in promoting coagulation. In vitro studies, it has found that each of endothelial cells, vascular smooth muscle cells, platelets, leucocytes, lymphocytes, erythrocytes, and the like are all able to release MVs. According to their source, MVs can be divided into two categories: exosomes and shedding vesicles. Exosomes are secreted in the manner of exocytosis with multi-vesicular bodies (MVBs) in the case of cells are stimulated, and shedding vesicles are directly secreted from the cell surface by budding. Presently, different names are given to shedding vesicles secreted by different cells, for example, those from neutrophil granulocytes and monocytes are called ectosomes, and those from platelets are called microparticles.
Membrane component of cellular MVs, depending on the cells from which they originate, is mainly composed of lipid and protein. However, the inner component of cellular MVs is still unknown. The plasma membrane of cellular MVs contains the features of its original cells, i.e. contains specific molecular markers and cell receptors/ligands on the surface of the original cells. Definite physiological functions of cellular MVs have not been investigated clearly up to now.
Interfering RNA (siRNA) is a kind of double-stranded RNA molecule consisting of more than 20 nucleotides, and it plays a role in silencing gene expression through the specific degradation of messenger RNA (mRNA). This process is called RNA interference (RNAi).
RNAi is a way of post-transcriptional gene silencing, and is one of the old and evolutive highly conserved phenomena in the living nature. Through siRNA mediated recognition and targeting cleavage of homologous target mRNA, gene expression is suppressed specifically and efficiently. RNA interference has the characteristics of biocatalytic reaction, in which multiple proteins and ATP were involved.
In recent years, study of RNA interference has made breakthroughs, has been rated as one of the top ten most scientific progress by the journal Science in 2001, and has been ranked the top ten most scientific progress in 2002. By using RNA interfering technology, the expression of the specific gene can be knocked out or turn off. Therefore, the RNA interfering technology has been widely used in the fields of bio-medical experiment research and gene therapy of various diseases.
Before the RNA interfering technology appearing, gene knockout is the major research tool in reverse genetics, but with high difficulty of technology, complex operation and long time of research. RNA interference has been now an important research tool for exploring the function of genes due to it could use siRNA or siRNA expression vector with faster, cheaper, simply and highly sequence-specific to silence the specific gene specifically to obtain the mutation sequence with lost or decreased function so as to knockout the expression of target gene specifically. In the study of functional genomes, specific gene needed to be functional-loss or mutation sequence reduction so as to confirm its function. Therefore, RNAi can be used to the study of functional genomes as a powerful study tool. Meanwhile, the establishment of the method for construction of siRNA expression library enables the high throughput screening using RNAi technology, it has important significance in both clarification of signal transduction pathway and discovery of new drug targets.
RNAi is also wildly used in the field of treatment of diseases. In the stud of gene therapy for HIV-1, Hepatitis B and Hepatitis C etc. using RNA interference technology, it is found that selecting sequence in the viral genome that has no homology to sequences in the human as the suppression sequence can void the side effect on the normal tissues while inhibiting the replication of virus. At the same time, choosing the suppression sequence at the special would induce apoptosis of some malignant cells with definite gene mutation. Moreover, tumor cells can be killed specifically by introducing the expression of siRNA or shRNA for some oncogenes or molecules against apoptosis using the promoters specific for tumors.
As RNA interference is the gene silencing against prost-transcriptional stage, corresponding to the gene knockout genetically with traditional gene therapy, RNAi is more simply in the whole process design, and the action is rapid and effect obviously, which opens a new way for the gene therapy. The general idea is that through strengthening the mechanism of RNA interference of the key gene, to control the abnormal progress of protein synthesis appearing in diseases and replication or expression of exogenous pathogenic nucleic acid, especially some nucleic acid viruses seriously harm to human health by strengthening the mechanism of RNA interference of the key gene.
In recently, studies have demonstrated that siRNA can inhibit the replication of HIV in the cells cultured in vitro. HIV infection could be prevented by siRNA through inhibit its own gene (e.g. pie, gag, rev, tat and env) of HIV virus and its host gene (e.g. CD4, major receptor of HIV). Meanwhile, studies have found that siRNA inhibiting Fas is injected intravenously into the mouse in two mouse models with autoimmune hepatitis, it is observed that Fas mRNA and protein level in liver cells is reduced, thus preventing liver cells from damages of apoptosis caused by autoimmune hepatitis. Moreover, studies have found that transformation of tumor cells from benign to malignant can be inhibited by silencing p53 gene through RNAi.
Although RNAi has been widely used in every aspects of bio-medical research, there are still some problems difficult to be solved. For example, the efficiency of transferring siRNA to some cells, e.g. immune cells, is very low using the existing transfection method of liposome, which will affect further application of it in this field.
Meanwhile, although many achievements were made in the research and development siRNA drugs, it still faces many problems for applying it into real medical treatment. Although siRNA can be directly injected into the animals, the half life of the siRNA without encapsulating is very short, and the therapeutic efficacy is barely satisfactory. Presently, the carriers of delivering siRNA drugs mainly include liposomes, nanocapsules/nanoparticles, β-cyclodextrin inclusion compound (or also called β-cyclodextrin capsule) and so on. These carriers can partly prolong the retention time of the drugs in vivo and increase the absorption rate siRNA drug, but the targeting and high efficiency of delivering drugs are still weak. Problems of how to effectively administration to human while ensuring the drug release of the efficacy at target tissues and organs as well as having higher safety and the like are all needed to further investigation.
As an important bio-medical research tool and a potential drug, siRNA is now facing some open problems, and the poor specificity (targeting), less stability and lower efficiency of delivering siRNA are the main reasons for limiting its use. Therefore, it is an urgent need for a more stable, high effective and specific way of delivering siRNA to deliver siRNA high effectively and specifically.
It is unexpectedly for the applicant that cellular MV is a vector of bio-vesicle vehicle with highly effective rate and specificity in vivo. These cellular MVs are variable in size, ranging between 10-500 nm. In principle, the membrane components (including specific surface receptors and membrane lipid structures) of MVs secreted by different cells are the same as the plasma membrane components of the corresponding cells. Therefore, cellular MVs carrying with receptor proteins or membrane lipid structure from the surface of the cells, have high affinity to the corresponding target cells. Using cellular MVs as a carrier for delivering siRNA, siRNAs can be selectively delivered into the target cells/tissues high efficiently and selectively, thus enhancing the regulation of cellular functions greatly. It is obviously that since the cellular MVs (including the membrane lipid vesicular structures with characteristics similar to the cellular MVs, such as exosomes and shedding vesicles as well as particular shedding vesicles secreted by different cells) themselves have the specificity of binding to particular tissues and cells, the siRNA carried by cellular MV also exhibit high targeting, stability and efficiency, they have a significant application prospect in the study and therapy of the mechanism of diseases.
The inventors of the present invention find that using the cellular MV as a vector to deliver the interfering RNA to the target cells will not harm to organisms themselves, due to the cellular MV are substances secreted by cells themselves and have bio-affinity; meanwhile, cellular MVs can be transferred into the target cells efficiently and selectively due to the surface of which carry surface molecules originating from cells and have high affinity to the target cells. The interfering RNA can functions by combined with specific sequence of target gene mRNA to block the translation process of protein of the target gene, thus playing a role blocking the gene expression specifically.
The advantages of using cellular MVs as a vector to deliver siRNA are: firstly, cellular MVs originate from cells, and is a native existence of organism, thus it can overcome the toxicity to cells and damage to the body of the drug carriers presently synthesized; secondly, various technical ways used during enclosing siRNA into cellular MVs are all easy to implement and the enclosing efficiency is very high, which increase its application potential in practical to a certain degree; more importantly, cellular MV are vesicle structures with a lipid bi-layer membrane and the structure of the outer membrane is similar to that of cytoplasm, which can enter the cell through fusion with the cell membrane and endocytosis. Meanwhile, cellular MVs would enter the target cells efficiently and selectively due to its surface carrying with molecule markers such as surface protein and various receptors/ligands originating from the surface of the cytoplasm of cells. If using the cellular MVs excreted from the primary culture of tissues or cells of patients themselves to enclose siRNA, immune rejection can be reduced and the transferring efficiency of the cellular MVs carrying siRNA to organism can be further improved. Based on the above-mentioned advantages, as a carrier to deliver the siRNA as a drug, cellular MVs will play a more important role in the development of drugs and prevention and treatment of the clinical diseases.